The primary immunologic abnormality resulting from infection by HIV is the progressive depletion and functional impairment of T lymphocytes expressing the CD4 cell surface glycoprotein (Lane et al., Ann. Rev. Immunol. 3:477, 1985). CD4 is a non-polymorphic glycoprotein with homology to the immunoglobulin gene superfamily (Maddon et al., Cell 42:93, 1985). Together with the CD8 surface antigen, CD4 defines two distinct subsets of mature peripheral T cells (Reinherz et al., Cell 19:821, 1980), which are distinguished by their ability to interact with nominal antigen targets in the context of class I and class II major histocompatibility complex (MHC) antigens, respectively (Swain, Proc. Natl. Acad. Sci. 78:7101, 1981; Engleman et al., J. Immunol. 127:2124, 1981; Spitz et al., J. Immunol. 129:1563, 1982; Biddison et al., J. Exp. Med. 156:1065, 1982; and Wilde et al., J. Immunol. 131:2178, 1983). For the most part, CD4 T cells display the helper/inducer T cell phenotype (Reinherz, supra), although CD4 T cells characterized as cytotoxic/suppressor T cells have also been identified (Thomas et al., J. Exp. Med. 154:459, 1981; Meuer et al., Proc. Natl. Acad. Sci. USA 79:4395, 1982; and Krensky et al., Proc. Natl. Acad. Sci. USA 79:2365, 1982). The loss of CD4 helper/inducer T cell function probably underlies the profound defects in cellular and humoral immunity leading to the opportunistic infections and malignancies characteristic of the acquired immunodeficiency syndrome (AIDS) (H. Lane supra).
Studies of HIV-I infection of fractionated CD4 and CD8 T cells from normal donors and AIDS patients have revealed that depletion of CD4 T cells results from the ability of HIV-I to selectively infect, replicate in, and ultimately destroy this T lymphocyte subset (Klatzmann et al., Science 225:59, 1984). The possibility that CD4 itself is an essential component of the cellular receptor for HIV-I was first indicated by the observation that monoclonal antibodies directed against CD4 block HIV-I infection and syncytia induction (Dalgleish et al., Nature (London) 312:767,1984; McDougal et al., J. Immunol. 135:3151, 1985). This hypothesis has been confirmed by the demonstration that a molecular complex forms between CD4 and gp120, the major envelope glycoprotein of HIV-I (McDougal et al., Science 231:382, 1986); and the finding that HIV-I tropism can be conferred upon ordinarily non-permissive human cells following the stable expression of a CD4 cDNA (Maddon et al., Cell 47:333, 1986).
The widespread use of highly active antiretroviral therapy (HAART) has dramatically improved the clinical course for many individuals infected with HIV (Berrey, M. M. et al., J Infect Dis 183(10):1466, 2001). However, toxicities associated with long term HAART have put a high priority on the design and development of less toxic therapies. Among the “next generation” of antiviral inhibitors is T-20 (Wild, C. et al., Proc Natl Acad Sci USA 91(26):12676, 1994; Wild, et al. Proc Natl Acad Sci USA 89(21):10537, 1992), a relatively non-toxic peptide that disrupts viral fusion thereby protecting CD4+ lymphocytes from de novo infection. In clinical trials T-20 has been shown to reduce plasma viral load by up to two logs (Kilby, et al., Nat Med 4(11):1302, 1998). These results demonstrate that the entry stage of the HIV replication cycle is a viable target for the development of new antiretroviral therapies.
Viral entry is a complex biochemical event that can be subdivided into at least three stages: receptor docking, viral-cell membrane fusion, and particle uptake (D'Souza, M.P. et al., Jama 284(2):215, 2000). Receptor docking is a multi-step process that begins with the gp120 component of a virion spike binding to the CD4 receptor on the target cell. Conformational changes in gp120 induced by gp120-CD4 interaction promote a high affinity interaction between gp120 and either CCR5 or CXCR4 cellular co-receptors. This is followed by gp41 mediated fusion of the viral and target cell membranes. Agents designed to block gp120-CD4, gp120-CCR5/CXCR4 or gp41/cell membrane interactions are in various stages of development (D'Souza, M. P. et al., Jama 284(2):215, 2000). Several laboratories have constructed recombinant fusion proteins that fuse the gp120 binding domain of CD4 to immunoglobulin constant domains (Deen, K. C. et al., Nature 331(6151):82, 1988; Fisher, R. A. et al., Nature 331(6151):76, 1988; Capon, D. J. et al., Nature 337(6207):525, 1989; Traunecker, A. et al., Nature 339(6219):68, 1989; Trkola, A. et al., J Virol 69(11):6609, 1995). One of these, Pro-542 is currently being evaluated in clinical trials (Jacobson, J. M. et al., J Infect Dis 182(1):326, 2000).
The strategy underlying these CD4 based therapies, i.e. blocking the interaction between gp120 and the CD4 receptor, encompasses advantages distinct from current HAART regimens. The CD4 binding site on gp120 includes highly conserved residues; thus, agents targeting this site are unlikely to encounter resistance mutants. Additionally, such agents, by blocking de novo infection, may prevent the expansion of viral reservoirs.
Monomeric soluble CD4 (sCD4) was one of the first reagents in this group to be tested clinically (Schooley et al., Ann Intern Med 112(4):247, 1990). Unfortunately, sCD4 failed to demonstrate significant antiviral activity in vivo (Schooley et al., Ann Intern Med 112(4):247, 1990). Among the problems inherent to sCD4 was its inability to efficiently neutralize primary isolates of HIV. The differential capacity of sCD4 to neutralize tissue culture laboratory adapted (TCLA) strains versus many primary isolates is striking. In the initial report describing this difference, Ho and colleagues found that the concentrations of sCD4 required to neutralize primary isolates were up to 1000-fold higher than those required to neutralize TCLA strains (Ashkenazi et al., Proc Natl Acad Sci USA 88(16):7056, 1991). Surprisingly, when the affinities of sCD4 for soluble gp120s derived from TCLA and primary isolates were measured, no correlation between sCD4 neutralization and CD4:gp120 affinity was observed (Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88(16):7056,1991; Brighty et al., Proc. Natl. Acad. Sci. USA 88(17):7802, 1991; Ivey-Hoyle et al., Proc. Natl. Acad. Sci. USA 88(2):512, 1991). However, the affinity of sCD4 for gp120 on primary virions was reduced relative to gp120 on the surface of TCLA virions (Moore et al., J Virol 66, 235-243, 1992). The basis for the differential interaction between sCD4 and soluble gp120 vs. virion associated gp120 is unclear.
There is an additional property of sCD4 that may, at least in part, explain its inability to neutralize primary isolates. At low concentrations sCD4 enhances the infectivity of most primary isolates (Moore et al., Aids 9, Suppl A:S117, 1995; Sullivan et al., J Virol 69(7):4413, 1995; Moore et al., J Virol 66(1):235, 1992; Orloff et al., J Virol 67(3):1461, 1993; Schutten et al., Scand J Immunol 41(1):18, 1995; Willey et al., J Virol 68(2):1029, 1994). Although these observations were made prior to the identification of the HIV fusion/coreceptors, several research groups suggested that sCD4 mediated enhancement resulted from the activation of the fusion component of virion associated spikes (Sullivan et al., J Virol 69(7):4413, 1995; Fu et al., J Virol 67(7):3818, 1993). As has since become clear, sCD4 engagement of gp120 results in conformational changes in gp120 that promote its interaction with CCR5 and thus initiates the process of virus-cell fusion (Doranz et al., J Virol 73(12):10346, 1999; Trkola et al., Nature 384(6605):184, 1996; Wu et al., Nature 384(6605):179, 1996; Zhang et al., Biochemistry 38(29):9405, 1999).
Because sCD4-mediated enhancement of virus infectivity is only observed at low concentrations of sCD4, it likely reflects a condition where virions bear a mixture of unoccupied gp120s along with sCD4-bound gp120s. Neutralization occurs only when the concentration of sCD4 reaches a threshold level where a sufficient number of spikes per virion are prevented from participating in the fusion process. The concentration required to achieve that state is likely to be extremely high for two reasons: 1) sCD4 must compete with surface bound CD4 receptors which are presented in bulk on the surface of a target cell and the effects of avidity strongly favor the receptors presented on the membrane. The lack of high avidity associated with monomeric sCD4 is a critical deficiency in the antiviral activity of this molecule, and 2) sCD4 promotes a high affinity interaction between gp120 and CCR5 (Doranz et al., J Virol 73(12):10346, 1999; Trkola, et al., Nature 384(6605):184, 1996; Wu et al., Nature 384(6605):179, 1996; Zhang et al., Biochemistry 38(29):9405, 1999). Thus, even at relatively high concentrations, sCD4 promotes interactions between the virion and the target cell membrane.
Regardless of the mechanism, it is clear that sCD4 is not the therapeutic agent of choice for treating HIV. Thus, a need remains for a CD4-based agent that can be used to study HIV infection in vitro, and is of use for treating or preventing HIV infection in vivo.